CHAPTER 14

Glucose Utilization and

Biosynthesis
Problems:5, 6, 9, 10, 13, 14, 17, 20, 22, and
26
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– Harnessing energy from glucose via
glycolysis
– Fermentation under anaerobic conditions
– Synthesis of glucose from simpler
compounds: gluconeogenesis
– Oxidation of glucose in pentose phosphate
pathway
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 Central Importance of
Glucose
• Glucose is an excellent fuel
– Yields good amount of energy upon oxidation
– Can be e!ciently stored in the polymeric form
– Many organisms and tissues can meet their energy
needs on glucose only
• Glucose is a versatile biochemical precursor
– Bacteria can use glucose supply metabolic
intermediates used to build the carbon skeletons
of:
• All the amino acids
• Membrane lipids
• Nucleotides in DNA and RNA
• Cofactors needed for the metabolism 2

2
 Four Major Pathways of
Glucose Utilization
• When there’s plenty of excess energy, glucose can be
stored in the polymeric form (starch, glycogen)
• Short-term energy needs are met by oxidation of
glucose via glycolysis
• Pentose phosphate pathway generates NADPH that is
used for detoxification, and for the reductive
biosynthesis of lipids and nucleotides and generates
Pentose phosphate
• Structural polysaccharides (e.g. in cell walls of bacteria,
fungi, and plants) are derived from glucose
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4

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 Glycolysis: Importance
• Glycolysis is a sequence of enzyme-catalyzed reaction
by which glucose is converted into pyruvate
• Pyruvate can be further aerobically oxidized (Citric Acid cycle
to yield carbon dioxide and water)
• Pyruvate can be used as a precursor in biosynthesis
• In the process, some of the oxidation free energy in
captured by the synthesis of ATP and NADH
• Research of glycolysis played a large role in the
development of modern biochemistry
– Understanding the role of coenzymes
– Discovery of the pivotal role of ATP
– Development of methods for enzyme purification
– Inspiration for the next generations of biochemists 5

5
 Glycolysis: Overview
• In the evolution of life, glycolysis probably was
one of the earliest energy-yielding pathways
• It developed before photosynthesis, when the
atmosphere was still anaerobic
• Thus, the task upon early organisms was how
to extract free energy from glucose
anaerobically?
•The solution
–Activate it first by transferring couple of
phosphates to it
–Collect energy later from the high-energy
metabolites of the activated glucose 6

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7

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Glycolysis: The Preparatory
Phase

8
For each molecule of glucose that passes through the
preparatory phase (a), two molecules of glyceraldehyde 3-
9
phosphate are formed
9
 Glycolysis: The Payo"
Phase
Pyruvate is the end product of the second phase of
glycolysis. For each glucose molecule, two ATP are
consumed in the preparatory phase and four ATP are
produced in the payoff phase, giving a net yield of two
ATP per molecule of glucose converted to pyruvate. The
numbered reaction steps are catalyzed by the enzymes
listed on the right. Keep in mind that each phosphoryl
group, represented here as P, has two negative charges
(—PO32–).

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Triose
phosphate
isomerase

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12

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 1.The Hexokinase Reaction
• The first step, phosphorylation of glucose, is
catalyzed by hexokinase in eukaryotes, and by
glucokinase in prokaryotes
• Nucleophilic oxygen at C6 of glucose attacks
the last (!) phosphorous of ATP
• Bound Mg++ facilitates this process by
stabilizing the negative charge in the
transition state
• This process uses the energy of ATP
• This process is irreversible
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 1.

Irreversible

Kinases catalyze phosphorylation of molecules by ATP.

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 2. Phosphohexose
Isomerization
• An aldose can isomerize into ketose via an
enediol intermediate
• The isomerization is catalyzed by the active-
site glutamate
• In one step, ionized Glu acts as a general
base to abstract the proton from C2 and
generate the enediol
• In the next step, protonated Glu acts as a
general acid to re-protonate enediol at C1
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 2.

Isomerases catalyze the transformation of compounds

into their positional isomers.

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 2. Mechanism of
Phosphohexose Isomerase

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2.

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2.

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2.

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3. The Second Priming Reaction;
The First Commitment
• ATP is the donor of the second phosphate group
• This is an irreversible step
• The product, fructose 1,6-bisphosphate is committed
to become pyruvate and yield energy
• Phosphofructokinase-1 is negatively regulated by
ATP
– Do not burn glucose if there is plenty of ATP
– This process is irreversible

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3.
Irreversible

!G’0 = -14.2 kJ/mol

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 4. Aldolases Cleave 6-
Carbon Sugars
• The reverse process is the familiar aldol
condensation
• Animal and plant aldolases employ covalent
catalysis
• Fungal and bacterial aldolases employ metal
ion catalysis
• Aldolases catalyze reversible aldol
condensation

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4

DAP GAP

"G’0 is positive but

the reactants are
present in low
concentrations.

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 5. Triose Phosphate
Interconversion
• Aldolase creates two triose phosphates: DAP
and GAP
• Only GAP is the substrate for the next
enzyme (Step 6)
• DAP is converted enzymatically to GAP

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5.

GAP is continuously used up.

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 6. Glyceraldehyde 3-
Phosphate Dehydrogenase
Reaction
• First energy-yielding step in glycolysis
• Oxidation of aldehyde with NAD+ gives
NADH
• Phosphorylation yields an high-energy
reaction product

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29

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6.

Very high energy of

hydrolysis:
-49.3 kJ/mol

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 7. First Substrate-Level
Phosphorylation
• 1,3-bisphosphoglycerate is a high-energy
compound that can donate the phosphate
group to ADP to make ATP
• The reaction is reversible, the reverse process
transfer of phosphate from ATP to
phosphoglycerate
• Kinases are enzymes that transfer phosphate
groups from molecules like ATP to various
substrates
• Substrate level phosphorylation 31

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7.

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 8. Conversion of 3-
Phosphoglycerate to 2-
Phosphoglycerate
• This is a reversible isomerization reaction
• Enzymes that shift functional groups
around are called mutases

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8.

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 8. Mechanism of the
Phosphoglycerate Mutase
Reaction
• Phosphoglycerate mutase employs covalent
catalysis
• One of the active site histidines is post-
translationally modified to phosphohistidine
• Phosphohistidine donates its phosphate to O2
before retrieving another phosphate from O3

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 8. Mechanism of the
Phosphoglycerate Mutase
Reaction
• Notice that the phosphate from the substrate
ends up bound to the enzyme at the end of
the reaction

• The two negative charges in the product are

fairly close now but 2-phosphoglycerate is
not good enough phosphate donor

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38

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 9. Dehydration of 2-
Phosphoglycerate
• The goal here is to create a better phosphoryl
donor
• Loss of phosphate from 2-phosphoglycerate
would merely give a secondary alcohol with no
further stabilization.

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9.

1 2

Energy to transfer phosphate from 1. = -17.6 kJ/mol

Energy to transfer phosphate from 2= -61.9 kJ/mol
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 10 Second Substrate-Level
Phosphorylation
• … but loss of phosphate from
phosphoenolpyruvate yields an enol that
tautomerizes into ketone
• The tautomerization e"ectively lowers the
concentration of the reaction product and
drives the reaction toward ATP formation
• Substrate level phosphorylation
• Irreversible reaction
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10

Irreversible

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43

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 Pyruvate Kinase is Subject
to Regulation
• Pyruvate kinase requires divalent metals
(Mg++ or Mn++) for activity
• Under physiological conditions, the activity
of pyruvate kinase is limited by the level of
Mg++
• When there is plenty of ATP, the Mg ions
are sequestered by ATP; this slows down
pyruvate kinase
• Increased concentration of metabolites in
the glycolytic pathway slows down glucose
utilization 44

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 Glycolysis Occurs at
Elevated Rates in Tumor
Cells

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46

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 The anaerobic metabolism of
glucose in tumor cells yields far
less ATP (2 per glucose) than the
glucose
transporters
complete oxidation to CO2 that
takes place in healthy cells under
aerobic conditions (~30 ATP per
glucose), so a tumor cell must
consume much more glucose to
produce the same amount of ATP.
Glucose transporters and most of
the glycolytic enzymes are
overproduced in tumors.
Compounds that inhibit
hexokinase, glucose 6-phosphate
dehydrogenase, or transketolase
block ATP production by
glycolysis, thus depriving the
cancer cell of energy and killing it.
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Glycolysis:
Glycolysis is a near-universal pathway by which a
glucose molecule is oxidized to two molecules of
pyruvate, with energy conserved as ATP and NADH.

All 10 glycolytic enzymes are in the cytosol, and all 10

intermediates are phosphorylated compounds of three or
six carbons.

In the preparatory phase of glycolysis, ATP is invested to

convert glucose to fructose 1,6-bisphosphate. The bond
between C-3 and C-4 is then broken to yield two
molecules of triose phosphate.

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In the payoff phase, each of the two molecules of
glyceraldehyde 3-phosphate derived from glucose
undergoes oxidation at C-1; the energy of this
oxidation reaction is conserved in the form of one
NADH and two ATP per triose phosphate oxidized.
The net equation for the overall process is

Glucose + 2NAD+ + 2ADP + 2Pi " 2 pyruvate +

2NADH + 2H+ 2ATP + 2H2O

Glycolysis is tightly regulated in coordination with

other energy-yielding pathways to assure a steady
supply of ATP.

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 Under Anaerobic Conditions,
Animals Reduce Pyruvate to
Lactate
• During strenuous exercise, lactate builds up in
the muscle
• The acidification of muscle prevents its
continuous strenuous work
• The lactate can be transported to liver and
converted to glucose there

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 Under Anaerobic Conditions,
Yeast Ferments Glucose to
Ethanol
• Both steps require cofactors
– Mg++ and thiamine pyrophosphate in
pyruvate decarboxylase
– Zn++ and NAD+ in alcohol dehydrogenase

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Mechanism of Aldehyde
Reduction by Alcohol
Dehydrogenase

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 Feeder Pathways for
Glycolysis
Other carbohydrates undergo glycolysis
when they are transformed into a
glycolytic intermediate.

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Endogenous Glycogen and Starch Are Degraded by
Phosphorolysis

Glycogen stored in animal tissues (primarily liver and

skeletal muscle), in microorganisms, or in plant
tissues can be mobilized for use within the same cell
by a phosphorolytic reaction catalyzed by glycogen
phosphorylase (starch phosphorylase in plants).

These enzymes catalyze an attack by Pi on the (#1"4)

glycosidic linkage that joins the last two glucose
residues at a nonreducing end, generating glucose 1-
phosphate and a polymer one glucose unit shorter.
Phosphorolysis preserves some of the energy of the
glycosidic bond in the phosphate ester glucose 1-
phosphate.
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Glucose 1-phosphate produced by glycogen
phosphorylase is converted to glucose 6-phosphate
by phosphoglucomutase, which catalyzes the
reversible reaction:
Glucose 1-phosphate glucose 6-phosphate 60

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In glycolysis it is an irreversible reaction:

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WORKED EXAMPLE 14-1 Energy Savings for Glycogen
Breakdown by Phosphorolysis. Calculate the energy
savings (in ATP molecules per glucose monomer)
achieved by breaking down glycogen by phosphorolysis
rather than hydrolysis to begin the process of glycolysis.

Solution: Phosphorolysis produces a phosphorylated

glucose (glucose 1-phosphate), which is then converted
to glucose 6-phosphate—without expenditure of the
cellular energy (1 ATP) needed for formation of glucose 6-
phosphate from free glucose. Thus only 1 ATP is
consumed per glucose monomer in the preparatory
phase, compared with 2 ATP when glycolysis starts with
free glucose. The cell therefore gains 3 ATP per glucose
monomer (4 ATP produced in the payoff phase minus 1
ATP used in the preparatory phase), rather than 2—a
saving of 1 ATP per glucose monomer.
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Feeder Pathways for Glycolysis

•Endogenous glycogen and starch, storage

forms of glucose, enter glycolysis in a two-
step process. Phosphorolytic cleavage of a
glucose residue from an end of the
polymer, forming glucose 1-phosphate, is
catalyzed by glycogen phosphorylase or
starch phosphorylase.
Phosphoglucomutase then converts the
glucose 1-phosphate to glucose 6-
phosphate, which can enter glycolysis.

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•Ingested polysaccharides and disaccharides are
converted to monosaccharides by intestinal
hydrolytic enzymes, and the monosaccharides
then enter intestinal cells and are transported to
the liver or other tissues.

•A variety of D-hexoses, including fructose,

galactose, and mannose, can be funneled into
glycolysis. Each is phosphorylated and
converted to glucose 6-phosphate, fructose 6-
phosphate, or fructose 1-phosphate.Conversion
of galactose 1-phosphate to glucose 1-phosphate
also occurs.

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 Gluconeogenesis: Precursors
for Carbohydrates
• Tissues that depend solely on glucose for
metabolic energy:
brain, nervous system, erythrocytes, testes, renal
medulla and embryonic tissue.

• Sometimes there is not enough glucose stored as

glycogen- then glucose is synthesized from non
carbohydrate precursors.

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 What Is Gluconeogenesis,
and How Does It Operate?
Synthesis of "new glucose" from common
metabolites
• Humans consume 160 g of glucose per
day
• 75% of that is in the brain
• Body fluids contain only 20 g of glucose
• Glycogen stores yield 180-200 g of
glucose
• So the body must be able to make its
own glucose
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The pathway from
phosphoenolpyruvate to glucose 6-
phosphate is common to the
biosynthetic conversion of many
different precursors of carbohydrates
in animals and plants. The path from
pyruvate to phosphoenolpyruvate
leads through oxaloacetate, an
intermediate of the citric acid cycle.
Any compound that can be
converted to either pyruvate or
oxaloacetate can therefore serve
as starting material for
gluconeogenesis. This includes
alanine and aspartate, which are
convertible to pyruvate and
oxaloacetate, respectively, and
other amino acids that can also
yield three- or four-carbon
fragments, the so-called
glucogenic amino acids (Table
14-4). Plants and photosynthetic
bacteria are uniquely able to convert
CO2 to carbohydrates

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We will focus on gluconeogenesis in
mammalian liver

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Glucogenic-
converted
into glucose

Ketogenic-
converted
into
ketones

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Substrates for Gluconeogenesis
in mammals
Pyruvate, lactate, glycerol, amino acids and
all TCA (tricarboxylic acid cycle)
intermediates can be utilized
• Fatty acids cannot!
• Why?
• Most fatty acids yield only acetyl-CoA
• Acetyl-CoA (through TCA cycle) cannot
provide for net synthesis of sugars

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The Central Relationship of
the Citric Acid Cycle to
Catabolism

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 CoA CO2
+ +
NAD+ NADH

Pyruvate acetyl-CoA

Pyruvate from glycolysis is converted

to acetyl-CoA Via Pyruvate
dehydrogenase. This is an Irreversible
reaction. The acetyl-CoA then goe into
the citric acid cycle.

Animals cannot get to pyruvate from

acetyl-CoA
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Opposing pathways of
glycolysis and
gluconeogenesis in rat liver.

The reactions of glycolysis

are on the left side, in red; the
opposing pathway of
gluconeogenesis is on the
right, in blue.
The major sites of regulation
of gluconeogenesis shown
here.

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 Gluconeogenesis

• Occurs mainly in liver and kidneys

• Not the mere reversal of glycolysis for 2
reasons:
– Energetics must change to make
gluconeogenesis favorable (delta G of
glycolysis = -74 kJ/mol, or about that)
– Reciprocal regulation: Gluconeogenesis
must turn one on and the other o".

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 Gluconeogenesis

• Seven steps of glycolysis are retained:

– Steps 2 and 4-9
• Three steps are replaced:
– Steps 1, 3, and 10 (the regulated
steps!)
• The new reactions provide for a
spontaneous pathway ("G negative in the
direction of sugar synthesis), and they
provide new mechanisms of regulation

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1
replaced

2
3
replaced

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 7

10
replaced

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Steps 1, 3, and 10 are replaced. These
are the non-reversible, regulated
steps. In cells these reactions have
large, negative free energy changes.
The other 7 reaction steps have a "G
near zero.

Steps 1, 3 and 10 are replaced by a

new set of reactions catalyzed by
enzymes. These reactions are
exergonic and irreversible in the
direction of glucose synthesis.
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 Synthesis of Oxaloacetate
• Conversion of pyruvate to energy-rich
phosphoenolpyruvate requires two energy-
consuming steps
• In the first step, pyruvate is transported into
mitochondria and converted into oxaloacetate
by pyruvate carboxylase

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10 -10B

-10A
"G’0 = -31.4 kJ/mol + 0.9 kJ/mol
"G = -16.7 kJ/mol - 25 kJ/mol

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 Pyruvate Carboxylase
Mitochondrial Enzyme
Pyruvate is converted to oxaloacetate -10A
• The reaction requires ATP and bicarbonate
as substrates
• Coenzyme Biotin is covalently linked to an
active site lysine
• Acetyl-CoA is an allosteric activator
• Regulation: when ATP or acetyl-CoA are
high, pyruvate enters gluconeogenesis
• Acetyl-CoA is produced by oxidation of fatty
acids and signals that fatty acids are
available as fuel. 82

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Acetyl-CoA and ATP signal that
energy is abundant.

Metabolites are converted into

glucose and maybe even into
glycogen.

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 In mitochondria,
pyruvate is converted
to oxaloacetate in a
biotin-requiring
reaction catalyzed by
pyruvate carboxylase.

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Pyruvate carboxylase is a
compartmentalized reaction. Pyruvate is
converted to oxaloacetate in the
mitochondria. Because oxaloacetate
cannot be transported across the
mitochondrial membrane, it must be
reduced to malate, transported to the
cytosol, and then oxidized back to
oxaloacetate ( -10B) before
gluconeogenesis can continue.

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 Oxaloacetate Picks Up
Phosphate from GTP
• The phosphoenolpyruvate carboxykinase
reaction occurs either in the cytosol or the
mitochondria.
- 10B

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In the cytosol, oxaloacetate is converted to
phosphoenolpyruvate by PEP carboxykinase. The CO2
incorporated in the pyruvate carboxylase reaction is lost here as
CO2. The decarboxylation leads to a rearrangement of electrons
that facilitates attack of the carbonyl oxygen of the pyruvate
moiety on the phosphate of GTP.
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Pyruvate + HCO3- + H+ # oxaloacetate + ADP + Pi

Oxaloacetate + NADH + H+ $ l-malate + NAD+

l-malate + NAD+ # oxaloacetate + NADH + H+

Oxaloactetate + GTP $ PEP + CO2 +GDP

Pyruvate + ATP + GTP + HCO3- # PEP + ADP + GDP + Pi

+CO2

"G’0 = + 0.9 kJ/mol

"G = - 25 kJ/mol

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 Fructose-1,6-
bisphosphatase
Hydrolysis of F-1,6-bisPase to F-6-P
• Thermodynamically favorable - "G in
liver is -8.6 kJ/mol ("G’0 = -16.3 kJ/
mol)
• Allosteric regulation:
– citrate stimulates
– fructose-2,6-bisphosphate inhibits
– AMP inhibits
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A high level of AMP indicates that the energy is low and
signals the need for ATP generation. Glycolysis is
needed.

Conversely, high levels of ATP and citrate indicate that

the energy is high and that biosynthetic intermediates
are abundant. Glycolysis is nearly switched off and
gluconeogenesis is promoted.

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3 -3

"G’0 = -16.3 kJ/mol

"G = -8.6 kJ/mol in

liver

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 Glucose-6-Phosphatase
Conversion of Glucose-6-P to Glucose
• Presence of G-6-Pase in ER of liver and
kidney cells makes gluconeogenesis
possible
• Muscle and brain do not undergo
gluconeogenesis
• G-6-P is hydrolyzed as it passes into the
ER
• ER vesicles filled with glucose di"use to
the plasma membrane, fuse with it and
open, releasing glucose into the
bloodstream.
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1 -1

" G’0 = -13.8 kJ/mol

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Glucose-6-phosphatase is localized in the endoplasmic reticulum
membrane. Conversion of glucose-6-phosphate to glucose occurs
during transport into the ER.

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The glucose-6-phosphatase reaction involves formation of
a phosphohistidine intermediate.

96
Glucose-6-phosphatase activity in controlled by the level of
glucose-6-phosphate. This is substrate level control.

Activity is not under allosteric control.

When levels of glucose-6-phosphate are high, glycolysis

switched off.

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Glycolysis:
Glycolysis is a near-universal pathway by which a
glucose molecule is oxidized to two molecules of
pyruvate, with energy conserved as ATP and NADH.

All 10 glycolytic enzymes are in the cytosol, and all 10

intermediates are phosphorylated compounds of three or
six carbons.

In the preparatory phase of glycolysis, ATP is invested to

convert glucose to fructose 1,6-bisphosphate. The bond
between C-3 and C-4 is then broken to yield two
molecules of triose phosphate.

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In the payoff phase, each of the two molecules of
glyceraldehyde 3-phosphate derived from glucose
undergoes oxidation at C-1; the energy of this
oxidation reaction is conserved in the form of one
NADH and two ATP per triose phosphate oxidized.
The net equation for the overall process is

Glucose + 2NAD+ + 2ADP + 2Pi " 2 pyruvate +

2NADH + 2H+ 2ATP + 2H2O

Glycolysis is tightly regulated in coordination with

other energy-yielding pathways to assure a steady
supply of ATP.

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Feeder Pathways for Glycolysis

•Endogenous glycogen and starch, storage

forms of glucose, enter glycolysis in a two-
step process. Phosphorolytic cleavage of a
glucose residue from an end of the
polymer, forming glucose 1-phosphate, is
catalyzed by glycogen phosphorylase or
starch phosphorylase.
Phosphoglucomutase then converts the
glucose 1-phosphate to glucose 6-
phosphate, which can enter glycolysis.

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•Ingested polysaccharides and disaccharides are
converted to monosaccharides by intestinal
hydrolytic enzymes, and the monosaccharides
then enter intestinal cells and are transported to
the liver or other tissues.

•A variety of D-hexoses, including fructose,

galactose, and mannose, can be funneled into
glycolysis. Each is phosphorylated and
converted to glucose 6-phosphate, fructose 6-
phosphate, or fructose 1-phosphate.Conversion
of galactose 1-phosphate to glucose 1-phosphate
also occurs.

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Gluconeogenesis

• Gluconeogenesis is a ubiquitous multistep

process in which glucose is produced from
lactate, pyruvate, or oxaloacetate, or any
compound (including citric acid cycle
intermediates) that can be converted to one of
these intermediates.

Seven of the steps in gluconeogenesis are

catalyzed by the same enzymes used in
glycolysis; these are the reversible reactions.

102
•Three irreversible steps in glycolysis are bypassed by
reactions catalyzed by gluconeogenic enzymes:
(1) conversion of pyruvate to PEP via oxaloacetate,
catalyzed by pyruvate carboxylase and PEP
carboxykinase;

(2) dephosphorylation of fructose 1,6-bisphosphate by

FBPase-1; and

(3) dephosphorylation of glucose 6-phosphate by

glucose 6-phosphatase.

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•Formation of one molecule of glucose from pyruvate
requires 4 ATP, 2 GTP, and 2 NADH; it is expensive.

•In mammals, gluconeogenesis in the liver, kidney, and

small intestine provides glucose for use by the brain,
muscles, and erythrocytes.

•Pyruvate carboxylase is stimulated by acetyl-CoA,

increasing the rate of gluconeogenesis when the cell has
adequate supplies of other substrates (fatty acids) for
energy production.

•Animals cannot convert acetyl-CoA derived from fatty acids

into glucose; plants and microorganisms can.

•Glycolysis and gluconeogenesis are reciprocally regulated

to prevent wasteful operation of both pathways at the same
time. 104

104
 Pentose Phosphate Pathway
• The main goals are to produce NADPH for
anabolic reactions and ribose 5-
phosphate for nucleotides

oxidations
NAD+ + 2e- + 2H+ # NADH + H+
NADP+ + 2e- + 2H+ # NADPH + H+
105
reductions
105
 Pentose Phosphate Pathway

• The main goals are to produce NADPH for

anabolic reactions and ribose 5-
phosphate for nucleotides

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Can Glucose Provide Electrons for
Biosynthesis?
Pentose Phosphate Pathway
aka hexose monophosphate shunt
• Provides NADPH for biosynthesis
• Produces ribose-5-P
• Two oxidative processes followed by
five non-oxidative steps
• Operates mostly in cytoplasm of liver
and adipose cells
• NADPH is used in cytosol for fatty acid
synthesis

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108

108
General scheme of the pentose phosphate pathway.

NADPH formed in the oxidative phase is used to reduce

glutathione, GSSG and to support reductive biosynthesis.
The other product of the oxidative phase is ribose 5-
phosphate, which serves as a precursor for nucleotides,
coenzymes, and nucleic acids.

In cells that are not using ribose 5-phosphate for

biosynthesis, the nonoxidative phase recycles six
molecules of the pentose into five molecules of the hexose
glucose 6-phosphate, allowing continued production of
NADPH and converting glucose 6-phosphate (in six cycles)
to CO2.

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109
Oxidative phase:
The end products are
ribose 5-phosphate,
CO2, and NADPH.

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110
Glucose-6-
phosphate is
oxidized:
The carbon
oxidation number
increased from +1 Equilibrium is
to +3. In favor of
NADPH
formation

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111
The lactone is
hydrolyzed to the free
acid
6-phosphogluconate
by a specific
lactonase.

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112
+2
6-phosphogluconate
undergoes oxidation
and decarboxylation
by 6-
phosphogluconate
dehydrogenase to
form the ketopentose
ribulose 5-phosphate;
the reaction
+4
generates a second
molecule of NADPH.

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114

114
 Phosphopentose isomerase converts ribulose 5-
phosphate to its aldose isomer, ribose 5-phosphate. In
some tissues, the pentose phosphate pathway ends at
this point, and its overall equation is:

The net result is the production of NADPH, a reductant for

biosynthetic reactions, and ribose 5-phosphate, a
precursor for nucleotide synthesis.

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The Nonoxidative Phase Recycles Pentose Phosphates
to Glucose 6-Phosphate:

In tissues that require primarily NADPH, the pentose

phosphates produced in the oxidative phase of the pathway
are recycled into glucose 6-phosphate. In this nonoxidative
phase, ribulose 5-phosphate is first epimerized to xylulose 5-
phosphate.

Epimers differ in configuration at one asymmetric center in

compounds that have two or more asymmetric centers.

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In a series of rearrangements of the carbon skeletons 6 five-
carbon sugar phosphates are converted to 5 six-carbon sugar
phosphates, completing the cycle and allowing continued
oxidation of glucose 6-phosphate with production of NADPH.
Continued recycling leads ultimately to the conversion of
glucose 6-phosphate to six CO2.
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Two enzymes unique to the pentose phosphate pathway act
in these interconversions of sugars: transketolase and
transaldolase. Transketolase catalyzes the transfer of a
two-carbon fragment from a ketose donor to an aldose
acceptor. Next, transaldolase removes a 3 carbon
fragment and condenses it with glyceraldehyde-3-
phosphate forminfg fructose-6-phosphate.

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 NADPH Regulates Partitioning
into Glycolysis vs. Pentose
Phosphate Pathway
• NADPH inhibits
glucose-6-phosphate
dehydrogenase

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Role of NADPH in
regulating the partitioning
of glucose 6-phosphate
between glycolysis and the
pentose phosphate
pathway. When NADPH is
forming faster than it is
being used for
biosynthesis and
glutathione reduction (see
Figure 14-20), [NADPH]
rises and inhibits the first
enzyme in the pentose
phosphate pathway. As a
result, more glucose 6-
phosphate is available for
glycolysis.

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Pentose Phosphate Pathway of Glucose Oxidation

•The oxidative pentose phosphate pathway

(phosphogluconate pathway, or hexose monophosphate
pathway) brings about oxidation and decarboxylation at C-1
of glucose 6-phosphate, reducing NADP+ to NADPH and
producing pentose phosphates.

•NADPH provides reducing power for biosynthetic reactions,

and ribose 5-phosphate is a precursor for nucleotide and
nucleic acid synthesis. Rapidly growing tissues and tissues
carrying out active biosynthesis of fatty acids, cholesterol, or
steroid hormones send more glucose 6-phosphate through
the pentose phosphate pathway than do tissues with less
demand for pentose phosphates and reducing power.

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•Entry of glucose 6-phosphate either into
glycolysis or into the pentose phosphate pathway
is largely determined by the relative
concentrations of NADP+ and NADPH.

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 Chapter 14:
Summary
In this chapter, we learned about:

• Glycolysis, a process by which cells can extract a

limited amount of energy from glucose under
anaerobic conditions
• Gluconeogenesis, a process by which cells can use a
variety of metabolites for the synthesis of glucose
• Pentose phosphate pathway, a process by which cells
can generate reducing power (NADPH) that is needed
for the biosynthesis of various compounds

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5. Energetics of the Aldolase Reaction

Aldolase catalyzes the glycolytic reaction:

Fructose 1,6-bisphosphate " glyceraldehyde 3-

phosphate + dihydroxyacetone phosphate

The standard free-energy change for this reaction in the

direction written is +23.8 kJ/mol. The concentrations of
the three intermediates in the hepatocyte of a mammal
are: fructose 1,6-bisphosphate,1.4 X10-5 M;
glyceraldehyde 3-phosphate, 3 X10-6 M; and
dihydroxyacetone phosphate, 1.6 X 10-5 M. At body
temperature (370 C), what is the actual free-energy
change for the reaction?

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4

DAP GAP

"G’0 is positive but

the reactants are
present in low
concentrations.

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127

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6. Pathway of Atoms in Fermentation
A “pulse-chase“ experiment using 14C-labeled carbon sources is
carried out on a yeast extract maintained under strictly anaerobic
conditions to produce ethanol.
The experiment consists of incubating a small amount of 14C-labeled
substrate (the pulse) with the yeast extract just long enough for each
intermediate in the fermentation pathway to become labeled. The
label is then “chased” through the pathway by the addition of excess
unlabeled glucose. The chase effectively prevents any further entry
of labeled glucose into the pathway.

(a)If [1-14C glucose (glucose labeled at C-1 with 14C) is used as a

substrate, what is the location of 14C in the product ethanol? Explain.

Figure 14–6 illustrates the fate of the carbon atoms of glucose. C-1
(or C-6) becomes C-3 of glyceraldehyde 3-phosphate and
subsequently pyruvate. When pyruvate is decarboxylated and
reduced to ethanol, C-3 of pyruvate becomes the C-2 of ethanol
(14CH3—CH2—OH).

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129

129
Triose
phosphate
isomerase

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131

131
(b) Where would 14C have to be located in the starting
glucose to ensure that all the 14C activity is liberated as
14CO during fermentation to ethanol? Explain.
2

If all the labeled carbon from glucose is converted to

14CO during ethanol fermentation,
2
the original label must have been on C-3 and/or C-4 of
glucose, because these are converted to the carboxyl
group of pyruvate.

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9. Equivalence of Triose Phosphates
14C -Labeled glyceraldehyde 3-phosphate was added to a yeast

extract. After a short time, fructose 1,6-bisphosphate labeled with

14C at C-3 and C-4 was isolated.

What was the location of the 14C label in the starting

glyceraldehyde 3-phosphate? Where did the second 14C label in
fructose 1,6-bisphosphate come from? Explain.

Problem 1 outlines the steps in glycolysis involving fructose 1,6-

bisphosphate, glyceraldehyde 3-phosphate, and
dihydroxyacetone phosphate. Keep in mind that the aldolase
reaction is readily reversible and the triose phosphate isomerase
reaction catalyzes extremely rapid interconversion of its
substrates. Thus, the label at C-1 of glyceraldehyde 3-phosphate
would equilibrate with C-1 of dihydroxyacetone phosphate (#G’0
=+7.5 kJ/mol). Because the aldolase reaction has #G’0 =- 23.8
kJ/mol in the direction of hexose formation, fructose 1,6-
bisphosphate would be readily formed, and labeled in C-3 and 133
C-4 (see Fig. 14–6).
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134
135

135
5.

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10. Glycolysis Shortcut Suppose you discovered a mutant yeast whose
glycolytic pathway was shorter because of the presence of a new
enzyme catalyzing the reaction:

Would shortening the glycolytic pathway in this way benefit the cell?
Explain.
Answer Under anaerobic conditions, the phosphoglycerate kinase and
pyruvate kinase reactions are essential. The shortcut in the mutant
yeast would bypass the formation of an acyl phosphate by
glyceraldehyde 3-phosphate dehydrogenase and therefore would not
allow the formation of 1,3-bisphosphoglycerate. Without the formation
of a substrate for 3-phosphoglycerate kinase, no ATP would be formed.
Under anaerobic conditions, the net reaction for glycolysis
normally produces 2 ATP per glucose. In the mutant yeast, net
production of ATP
would be zero and growth could not occur. Under aerobic conditions,
however, because the majority of ATP formation occurs via oxidative
phosphorylation, the mutation would have no observable effect.
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13. Free-Energy Change for Triose Phosphate Oxidation

The oxidation of glyceraldehyde 3-phosphate to 1,3-

bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate
dehydrogenase, proceeds with an unfavorable equilibrium constant
(K’eq 0.08; !G’0 =+ 6.3 kJ/mol), yet the flow through this point in the
glycolytic pathway proceeds smoothly. How does the cell overcome
the unfavorable
equilibrium?

Answer In organisms, where directional flow in a pathway is required,

exergonic reactions are coupled to endergonic reactions to overcome
unfavorable free-energy changes. The endergonic glyceraldehyde 3-
phosphate dehydrogenase reaction is followed by the
phosphoglycerate kinase reaction, which rapidly removes the product
of the former reaction. Consequently, the dehydrogenase reaction
does not reach equilibrium and its unfavorable free-energy change is
thus circumvented. The net !G’0 of the two reactions, when coupled,
is -18.5 kJ/mol=+ 6.3 kJ/mol =- 12.2 kJ/mol.
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6.

Very high energy of

hydrolysis:
-49.3 kJ/mol

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7.

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14. Arsenate Poisoning
Arsenate is structurally and chemically similar to inorganic
phosphate (Pi), and many enzymes that require phosphate will also
use arsenate. Organic compounds of arsenate are less stable than
analogous phosphate compounds, however. For example, acyl
arsenates decompose rapidly by hydrolysis:

On the other hand, acyl phosphates, such as 1,3-

bisphosphoglycerate, are more stable and undergo further enzyme-
catalyzed transformation in cells.
(a)Predict the effect on the net reaction catalyzed by glyceraldehyde
3-phosphate dehydrogenase if phosphate were replaced by
arsenate.
In the presence of arsenate, the product of the glyceraldehyde 3-
phosphate dehydrogenase reaction is 1-arseno-3-phosphoglycerate,
which nonenzymatically decomposes to 3- phosphoglycerate and
arsenate; the substrate for the phosphoglycerate kinase is therefore142
bypassed
142
(b) What would be the consequence to an organism if
arsenate were substituted for phosphate? Arsenate is very
toxic to most organisms. Explain why.

No ATP can be formed in the presence of arsenate

because 1,3-bisphosphoglycerate is not formed. Under
anaerobic conditions, this would result in no net glycolytic
synthesis of ATP. Arsenate poisoning can be used as a
test for the presence of an acyl phosphate intermediate in
a reaction pathway.

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17. Synthesis of Glycerol Phosphate
The glycerol 3-phosphate required for the synthesis of
glycerophospholipids can be synthesized from a glycolytic
intermediate. Propose a reaction sequence for this conversion.
Answer Glycerol 3-phosphate and dihydroxyacetone-3-phosphate
differ only at C-2. A dehydrogenase with the cofactor NADH acting
on dihydroxyacetone-3-phosphate would form glycerol-3-phospate.

In fact, the enzyme glycerol 3-phosphate

dehydrogenase catalyzes this reaction (see
Fig. 21–17).
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20. Pathway of Atoms in Gluconeogenesis
A liver extract capable of carrying out all the normal
metabolic reactions of the liver is briefly incubated in
separate experiments with the following 14C-labeled
precursors:

Trace the pathway of each precursor through gluconeogenesis.

Indicate the location of 14C in all intermediates and in the product,
glucose.
Answer
(a) In the pyruvate carboxylase reaction, 14CO2 is added to pyruvate
to form [4-14C]oxaloacetate, but the phosphoenolpyruvate
carboxykinase reaction removes the same CO2 in the next step. 145
Thus, 14C is not (initially) incorporated into glucose.
145
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146
 In mitochondria,
pyruvate is converted
to oxaloacetate in a
biotin-requiring
reaction catalyzed by
pyruvate carboxylase.

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148
22. Relationship between Gluconeogenesis and
Glycolysis
Why is it important that gluconeogenesis is not the
exact reversal of glycolysis?

Answer If gluconeogenesis were simply the reactions

of glycolysis in reverse, the process would be
energetically unfeasible (highly endergonic), because
of the three reactions with large, negative standard
free-energy changes in the catabolic (glycolytic)
direction.

Furthermore, if the same enzymes were used for all

reactions in the two pathways, it would be impossible
to regulate the two processes separately; anything that
stimulated (or inhibited) the forward reaction for a given
enzyme would stimulate (or inhibit) the reverse reaction
to the same extent. 149

149
26. Blood Lactate Levels during Vigorous Exercise The
concentrations of lactate in blood plasma before, during, and
after a 400 m sprint are shown in the graph.

(a)What causes the rapid rise in lactate concentration?

Rapid depletion of ATP during strenuous muscular exertion
causes the rate of glycolysis to increase dramatically, producing
higher cytosolic concentrations of pyruvate and NADH; lactate
dehydrogenase converts these to lactate and NAD+ (lactic acid
fermentation).
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151

151
(b) What causes the decline in lactate concentration after
completion of the sprint? Why does the decline occur
more slowly than the increase?
When energy demands are reduced, the oxidative
capacity of the mitochondria is again adequate, and
lactate is transformed to pyruvate by lactate
dehydrogenase, and the pyruvate is converted to
glucose. The rate of the dehydrogenase reaction is
slower in this direction because of the limited availability
of NAD and because the equilibrium of the reaction is
strongly in favor of lactate (conversion of lactate to
pyruvate is energy requiring).

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(c) Why is the concentration of lactate not zero during the
resting state?

The equilibrium of the lactate dehydrogenase reaction

is strongly in favor of lactate. Thus, even at very low
concentrations of NADH and pyruvate, there is a
significant concentration of lactate.

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